The adult human heart fails to repopulate the myocardium with sufficient new cardiomyocytes following injury, and this limited capacity for regeneration can lead to systolic heart failure. In contrast, organisms from diverse taxa including adult salamanders and zebrafish have remarkable abilities to achieve complete regeneration of the myocardium following heart injury. We collaborated with multiple other laboratories to perform an unbiased genomic screen to identify genes that are differentially expressed during heart regeneration in three well-described model systems with enhanced regenerative capabilities: zebrafish, axolotl (Ambystoma mexicanum, or the Mexican salamander) and neonatal mice. We identified the gene for the complement 5a receptor, C5aR1, to be induced in the regenerating heart within the first 48 hours following injury in all of these species, and further showed that this increase in expression is primarily localized to cardiomyocytes and endothelial cells. C5aR1 is a G-protein coupled receptor that is one of the key receptors activated by the C5a peptide of the complement cascade. The complement pathway is an ancient and highly conserved immune response system that can activate inflammation and promote clearance of foreign and damaged materials. When the complement system is activated, a series of sequential cleavage and downstream activation steps occur, leading to amplification and eventually a common pathway activation of multiple G-protein coupled receptors, including C5aR1, the focus of this project. Here we show preliminary data demonstrating that inhibition of the C5aR1 receptor following cardiac injury impairs the cardiomyocyte cell cycle response to apical resection in axolotl as well as in the neonatal mouse, suggesting that complement activation and signaling could represent an evolutionarily conserved pathway in successful heart regeneration. As described in this proposal, this project aims to reveal the mechanism of how the activated complement pathway promotes heart regeneration. Our central theory is that the complement pathway plays an evolutionarily-conserved role in the heart regenerative response by orchestrating molecular signaling between acute injury and cardiomyocyte proliferation, a crucial component of heart regeneration. The project has three hypothesis-drive mechanistic aims, focusing on neonatal mice as the mammalian heart regeneration system. Understanding the early events in successful heart regeneration may allow us to intervene rationally to promote regeneration in the acutely injured adult human heart, such as in the first days after a sizable infarction.